Identification, Optimization And Use Of Cryptic HLA-B7 Epitopes For Immunotherapy

ABSTRACT

The invention provides methods for identifying a HLA-B*0702-restricted cryptic epitope in an antigen, as well as methods for increasing the immunogenicity of HLA-B*0702-restricted cryptic epitopes. The HLA-B*0702-restricted cryptic epitopes and their cognate immunogenic epitopes are useful for stimulating an immune reaction against the cryptic epitopes in a subject. Accordingly, the invention further provides pharmaceutical compositions comprising a HLA-B*0702-restricted cryptic epitope or a cognate immunogenic epitope thereof, and vaccination kits comprising such epitopes. The novel materials of the invention are particularly useful for efficiently treating patients having an HLA-B*0702 phenotype.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/373,408, filed Jul. 9, 2009, which is a national stage applicationfiled under 35 U.S.C. 371 of International Application No.PCT/IB2007/003054, filed Jul. 12, 2007, which claims priority fromInternational Application No. PCT/IB2006/002937, filed Jul. 12, 2006,each of which is incorporated herein by reference in its entirety.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING SUBMITTED IN COMPUTER READABLE FORMAT

The Sequence Listing written in the file 433674SEQLIST.txt is 14,722bytes, and was created on Jun. 14, 2013, for the application filedherewith, Kosmatopoulos et al. “Identification, Optimization and Use ofCryptic HLA-B7 Epitopes for Immunotherapy.” The information contained inthis file is hereby incorporated by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to the field of peptide immunotherapy. Inparticular, the invention provides novel methods and materials forefficiently treating patients having an HLA-B*0702 phenotype.

Immunotherapy is a therapeutic approach which is currently the subjectof a great deal of interest in the context of the treatment of cancer.The principle thereof is based on immunization with peptides whichreproduce T cell epitopes of tumor antigens recognized by cytotoxic Tlymphocytes (CTLs) which play a major role in the elimination of tumorcells.

It will be recalled that CTLs do not recognize whole protein antigens,but peptide fragments thereof, generally comprising 8 to 11 amino acids,presented by class I major histocompatibility complex (MHC I) moleculesexpressed on the surface of cells. The presentation of these peptides isthe result of the antigen processing which involves three steps:

-   -   cytosolic degradation of the antigen by a multienzyme complex        called proteasome translocation of the peptides derived from        this degradation in the endoplasmic reticulum (ER) by the TAP        transporters    -   association of these peptides with the MHC I molecules and        exportation of the peptide/MHC I complexes to the cell surface

The peptide/MHC I complexes interact with the specific T cell receptor(TCR) on CTL inducing the stimulation and amplification of these CTLwhich become able to attack target cells expressing the antigen fromwhich the peptide is derived.

During the antigen processing, a peptide selection takes place, whichresults in a hierarchy of peptides presentation. Peptides that arepreferentially presented by the MHC I molecules are calledimmunodominant while peptides that are weakly presented are calledcryptic. Immunodominant peptides exhibit a high affinity for the MHC Iand are immunogenic while cryptic peptides exhibit a low affinity forMHC I and are non-immunogenic.

Immunodominant peptides have widely been targeted by tumor vaccines inpreclinical and clinical studies with disappointing results (Bowne etal., 1999; Colella et al., 2000; Gross et al., 2004; Hawkins et al.,2000; Naftzger et al., 1996; Overwijk et al., 1998; Vierboom et al.,1997; Weber et al., 1998).

Tumor antigens are frequently self proteins over-expressed by tumors andexpressed at lower levels by normal cells and tissues. Immune system isunable to react against these self antigens because of the selftolerance process. Self-tolerance concerns mainly the immunodominantpeptides (Cibotti et al., 1992; Gross et al., 2004; Hernandez et al.,2000; Theobald et al., 1997) thus explaining the incapacity of thesepeptides to induce a tumor immunity.

Cryptic peptides are much less involved in self tolerance process(Anderton et al., 2002; Boisgérault et al., 2000; Cibotti et al., 1992;Friedman et al., 2004; Gross et al., 2004; Moudgil et al., 1999;Overwijk et al., 2003; Sinha et al., 2004) and can therefore induce anefficient tumor immunity providing their immunogenicity is enhanced(Disis et al., 2002; Dyall et al., 1998; Engelhorn et al., 2006; Grosset al., 2004; Grossmann et al., 2001; Lally et al., 2001; Moudgil andSercarz, 1994a; Moudgil and Sercarz, 1994b; Palomba et al., 2005).

The usual strategy for enhancing the immunogenicity of cryptic peptides,that because of their low MHC I affinity are non-immunogenic, consistsin increasing their affinity for the MHC I molecules via amino acidssubstitutions. Peptide affinity for MHC I molecules mainly depends onthe presence at well defined positions (primary anchor positions) ofresidues called “primary anchor residues”. These residues are MHC Iallele specific. The presence of primary anchor residues although oftennecessary is not sufficient to ensure a high MHC I affinity. It has beenshown that residues located outside the primary anchor positions(secondary anchor residues) may exert a favourable or unfavourableeffect on the affinity of the peptide for the MHC I (Parker et al.,1994; Rammensee H et al., 1999). The presence of these secondary anchorresidues makes it possible to explain the existence, within the peptideshaving the primary anchor motifs, of a great variability in the bindingaffinity.

Amino acids substitutions aiming at enhancing affinity for MHC Imolecule should preserve the antigenicity of such optimized peptides.CTL generated by optimized peptides should cross-react with thecorresponding native peptides.

Many teams have succeeded in further enhancing immunogenicity of alreadyimmunogenic peptides by increasing their affinity for HLA-A*0201 (Bakkeret al., 1997; Parkhurst et al., 1996; Sarobe et al., 1998; Valmori etal., 1998). The inventors have previously described a general strategyto enhance affinity and immunogenicity of HLA-A*0201 restricted crypticpeptides (Scardino et al., 2002; Tourdot et al., 2000).

HLA-B*0702 is a frequently expressed molecule (25% of the population).Identification and optimization of HLA-B*0702 restricted tumor crypticpeptides should therefore be necessary in order to develop efficientcancer vaccines for HLA-B*0702 expressing patients.

Few tumor peptides presented by HLA-B*0702 have been described to date.Two peptides derived from the CEA (CEA₆₃₂) (Lu et al. 2000) and TERT(TERT₁₁₂₃) (Cortez-Gonzales et al. 2006) antigens have been identified;these peptides exhibited a strong binding affinity for HLA-B*0702 andwere immunogenic both in HLA-B*0702 transgenic mice and in vitro testswith human cells. These experimental results show that these peptidesare immunodominant peptides.

Two additional peptides derived from MAGE-A1 (MAGE-A1₂₈₉) (Luiten etal., 2000) and RU2 (a new antigen expressed by renal cell carcinoma)(Van den Eynde et al. 1999) have been identified to be targets ofHLA-B*0702 CTL that had been isolated from cancer patients. Althoughthere is no information about the HLA-B*0702 affinity of these twopeptides, we can consider them immunodominant because CTL developed incancer patients are always directed against immunodominant peptides.

SUMMARY OF THE INVENTION

As described in the experimental part below, the inventors have nowfound a general strategy to enhance affinity and immunogenicity of aHLA-B*0702 restricted cryptic peptide.

In a first aspect, the present invention provides a method forincreasing the immunogenicity of a MA-B*0702-restricted cryptic epitope,comprising a step of substituting the N-terminal residue of said epitopewith an alanine (A), or substituting the C-terminal residue of saidepitope with a leucine (L).

In what follows, the phrases “HLA-B*0702-restricted cryptic epitope” isused to designate a peptide, having 8 to 11 amino acids, more preferably9 or 10 amino acids, which exhibits a low affinity for HLA-B*0702, isnon immunogenic and has the sequence X₁PX₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁ (SEQ ID NO:58), wherein P is for proline, X₃ is R (arginine) or K (lysine) or H(histidine) or M (methionine), X₁ and X₄ to X₇ are independently anyamino acid, X₈ to X₁₀ are independently any amino acid or none, and theC-terminal amino acid X₁₁ is any amino acid with the proviso that if theN-terminal amino acid X₁ is A (alanine) then X₁₁ is neither L (leucine)nor A, nor I (isoleucine), nor V (valine), and nor M, and if X1 is anamino acid other than A then X₁₁ is L or A or I or V or M.

In the present text, the term “peptide” designates not only molecules inwhich amino acid residues are joined by peptide (—CO—NH—) linkages, butalso synthetic pseudopeptides or peptidomimetics in which the peptidebond is modified, especially to become more resistant to proteolysis,and provided their immunogenicity is not impaired by this modification.

In the present text, the amino acid residues are designated by their oneletter codes.

As used herein, the word “substituting” is to be understood as obtaininga peptide, the sequence of which is derived from the sequence of saidMA-B*0702-restricted cryptic epitope by the mentioned substitution, whenthe amino acid sequence of said cryptic epitope does not contain theappropriate amino acid, whatever the technical method used to obtainsaid peptide. For example, the peptide can be produced by artificialpeptide synthesis or by recombinant expression.

The affinity of a peptide for HLA-B*0702 can be determined by methodsknown in the art, for instance by the assay described by Rohrlich etal., 2003. Results are expressed as relative affinity (RA) when comparedto a reference peptide. Following this method a peptide is said to havea low affinity for HLA-B*0702 when RA is greater than 10. Peptides withRA greater than 10 are therefore considered to be cryptic peptides (orepitopes).

As used herein, the term “non immunogenic” refers to a peptide unable toinitiate a HLA-B*0702-restricted CTL immune response when administeredto a subject expressing HLA-B*0702 (including a HLA-B*0702 transgenicanimal).

In another embodiment, the immunogenicity of a HLA-B*0702-restrictedcryptic epitope in which the second and third amino acid residues are PRor PK or PH or PM and the last residue is L or A or I or V or M, can beincreased by substituting its first amino-acid by an A (alanine).Indeed, when the sequence of the selected HLA-B*0702-restricted crypticepitope is X₁PX₃X₄X₅X₆X₇X₈X₉ X₁₀X₁₁ (SEQ ID NO: 59), wherein theN-terminal amino acid X₁ is any amino acid but A, X₃ is R or K or H orM, the C-terminal amino acid X₁₁ is L or A or I or V or M, X₄ to X₇ areindependently any amino acid, and X₈ to X₁₀ are independently any aminoacid or none, the substitution of X₁ by A is sufficient to increase itsimmunogenicity.

In yet another embodiment, the immunogenicity of HLA-B*0702-restrictedcryptic epitopes in which the three first amino acid residues are APX₃(wherein X₃ is R or K or H or M) can be increased by substituting itslast amino-acid by a L (or by adding a leucine at its C-terminus,provided the amino acid sequence of said epitope after having added theleucine is not longer than 11 amino acids). Indeed, when the sequence ofthe selected HLA-B*0702-restricted cryptic epitope isAPX₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁ (SEQ ID NO: 60), wherein X₃ is R or K or H or M,X₄ to X₇ are independently any amino acid, X₈ to X₁₀ are independentlyany amino acid or none, and the C-terminal amino acid X₁₁ is an aminoacid other than L or A or I or V or M, the substitution of X₁₁ by L issufficient to increase its immunogenicity.

In what follows, the expression “optimized peptide” designates animmunogenic peptide derived from a HLA-B*0702-restricted cryptic epitopeby the above methods, and having the general sequenceAPX₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁ (SEQ ID No: 61), wherein X₃ is R or K or H or M,X₄ to X₇ are independently any amino acid, X₈ to X₁₀ are independentlyany amino acid or none, and the C-terminal amino acid X₁₁ is L or A or Ior V or M.

The inventors have identified a number of HLA-B*0702-restricted crypticepitopes, some of them are disclosed in Table I below. Accordingly,another aspect of the present invention is a crypticHLA-B*0702-restricted epitope, selected amongst the peptides of SEQ IDNOs: 1 to 4, disclosed in Table I.

TABLE I Selected cryptic HLA-B*0702 restricted peptides Antigen PeptideSEQ ID NO: TERT₄₄₄ DPRRLVQLL 1 HER-2/neu APRSPLAPS 2 SPKANKEIL 3GPKHSDCLA 4

Examples of immunogenic HLA-B*0702-restricted epitopes obtainedaccording to the present invention are those derived from a crypticHLA-B*0702-restricted epitope SEQ ID NOs 1, 3, 4, by substitution oftheir N-terminal amino-acid with an A (alanine) and those derived fromthe cryptic HLA-B*0702-restricted epitope SEQ ID NOs: 2 by substitutionof their C-terminal amino-acid with a L (leucine).

Therefore, the present invention also pertains to optimized peptidesderived from the cryptic peptides of SEQ ID NOs: 1 to 4, by a methodaccording to the invention. Preferred examples of optimized peptides areAPRSPLAPL (SEQ ID NO: 6), APKANKEIL (SEQ ID NO: 7), APKHSDCLA (SEQ IDNO: 8) and APRRLVQLL (SEQ ID NO: 5).

The invention also concerns a chimeric polypeptide, comprising two,three or more HLA-B*0702-restricted cryptic epitopes or two, three ormore immunogenic HLA-B*0702-restricted epitopes as described above. In achimeric polypeptide according to the invention, the epitopes can bedifferent from each other, or the same epitope can be repeated severaltimes (two, three or more times). The skilled artisan can chose anyknown technique to produce such polypeptides. For example, thepolypeptide can be obtained by chemical synthesis, or by using thetechnology of genetic engineering.

Another object of the present invention is an isolated nucleic acidmolecule designed to cause the expression of a crypticHLA-B*0702-restricted epitope or an immunogenic epitope or a chimericpolypeptide as above-described. By “designed to cause the expression of”a peptide is herein meant that said peptide is expressed as such,isolated from the whole antigen from which its sequence has beenselected (and, in appropriate cases, optimized as above-described), whenthe nucleic acid is introduced in an appropriate cell. The encodingregion for the epitope or chimeric polypeptide will typically besituated in the polynucleotide under control of a suitable promoter.Bacterial promoters will be preferred for expression in bacteria, whichcan produce the polypeptide either in vitro, or, in particularcircumstances, in vivo. An example of bacterium that can be used toproduce a peptide or polypeptide according to the invention, directly invivo, is Listeria monocytogenes, which is a facultative intracellularbacterium that enters professional antigen-presenting cells by activephagocytosis (Paterson and Maciag, 2005). Alternatively, a nucleic acidaccording to the invention can be administered directly, using anappropriate vector. In this case, a tissue-specific, a strongconstitutive, or an endogenous promoter can be used to control thepeptide expression. Suitable vector systems include naked DNA plasmids,liposomal compositions to enhance delivery, and viral vectors that causetransient expression. Exemplary are adenovirus or vaccinia virus vectorsand vectors of the herpes family, especially in a non-replicative form.

Another embodiment of the present invention is a pharmaceuticalcomposition comprising at least, as an active principle, aHLA-B*0702-restricted cryptic epitope as above-described, or animmunogenic epitope polypeptide derived therefrom as mentioned above, ora chimeric polypeptide according to the invention, or a nucleic acidencoding any of these, and/or a vector carrying said nucleic acid.Formulation of pharmaceutical compositions will accord with contemporarystandards and techniques. Medicines intended for human administrationwill be prepared in adequately sterile conditions, in which the activeingredient(s) are combined with an isotonic solution or otherpharmaceutical carrier appropriate for the recommended therapeutic use.Suitable formulations and techniques are generally described in thelatest edition of Remington's Pharmaceutical Sciences (Maack PublishingCo, Easton Pa.).

In particular, a HLA-B*0702-restricted cryptic epitope, or animmunogenic epitope polypeptide derived therefrom, or a chimericpolypeptide carrying several such immunogenic or cryptic epitopes, or anucleic acid encoding any of these, included or not in a vector, can beused for the preparation of a composition for preventive or curativeimmunotherapy, especially, for antiviral or anti-cancer immunotherapy.

In a particular embodiment, a pharmaceutical composition according tothe invention is a vaccine. In this latter case, the compositions ofthis invention can be combined with an adjuvant to potentiate the immuneresponse. Classic adjuvants include oil emulsions, like IncompleteFreund's Adjuvant, and adherent surfaces such as alum. Adjuvants thatrecruit and activate dendritic cells particularly via TLR (such asbacterial DNA or bacterial membrane derived proteins) or help to elicitcytotoxic T cells are especially useful. Other factors that otherwiseboost the immune response or promote apoptosis or elimination of cancercells can also be included in the composition.

Multiple doses and/or different combinations of the immunogeniccompositions of this invention can be packaged for distributionseparately or together. Each composition or set of compositions, such asthe kits of parts described below, can be accompanied with writteninstructions (in the form of promotional material or a package insert)regarding the use of the composition or combination in eliciting animmune response and/or the treatment of cancer.

In a previous patent application (PCT/EP2006/005325), the Applicant hasdescribed a vaccination protocol which enables the initiation andmaintenance of a T cell response targeting cryptic epitopes. The resultsreported in PCT/EP2006/005325 demonstrate that injection of a nativepeptide corresponding to a cryptic epitope, following vaccination withits cognate optimized peptide, can maintain the immune responseinitiated by said optimized peptide.

According to the invention, a HLA-B*0702-restricted cryptic epitope canhence be used for the preparation of a medicinal composition formaintaining the CTL immune response initiated by its cognate optimizedpeptide. An immunogenic peptide having an optimizedHLA-B*0702-restricted epitope sequence derived from aHLA-B*0702-restricted cryptic epitope can also be used, for thepreparation of a medicinal composition for initiating a CTL immuneresponse against said HLA-B*0702-restricted cryptic epitope. The presentinvention also encompasses a method for vaccinating a patient against atumoral or viral antigen, wherein said method comprises a first step ofvaccination with an optimized peptide cognate to a nativeHLA-B*0702-restricted cryptic epitope of said antigen, followed by asecond step of vaccination with said native peptide. In such a method,the first step and/or the second step can be performed by using achimeric polypeptide comprising two, three or more optimized or crypticpeptides as above-described, instead of single-epitope peptides.

The invention also pertains to a kit of parts comprising, in separateformulations, a first peptide having the sequence of aHLA-B*0702-restricted cryptic epitope, and a second peptidecorresponding to its cognate HLA-B*0702-restricted immunogenic epitope.Examples of peptides which can be part of a kit according to theinvention are the peptides of SEQ ID NOs: 1 to 4, which can constitutethe first peptide, the second peptide being then derived from said firstpeptide by a method for increasing its immunogenicity, as describedabove.

Other kits of parts according to the invention comprise at least achimeric polypeptide. Several variants of such kits are contemplated: ina first embodiment, the kit comprises, in separate formulations, a firstchimeric polypeptide comprising two, three or more HLA-B*0702-restrictedcryptic epitopes, and a second chimeric polypeptide corresponding to itscognate HLA-B*0702-restricted immunogenic chimeric polypeptide (whichmeans that it comprises optimized HLA-B*0702-restricted immunogenicepitopes cognate to the cryptic epitopes comprised in the first chimericpolypeptide). In a second embodiment, the kit comprises a first chimericpolypeptide comprising two, three or more HLA-B*0702-restricted crypticepitopes and, in one or several other separate formulations, peptidescorresponding to the optimized HLA-B*0702-restricted immunogenicepitopes cognate to the cryptic epitopes comprised in the first chimericpolypeptide. In a third embodiment, the kit comprises two, three or morepeptides corresponding to distinct HLA-B*0702-restricted crypticepitopes, wherein said peptides are either mixed in one singleformulation, or separated in several formulations and, in a separateformulation, a chimeric polypeptide comprising the optimizedHLA-B*0702-restricted immunogenic epitopes cognate to said crypticpeptides.

In the following description of the kits according to the invention,mention will be made only of the peptides (native or optimized), itbeing understood that chimeric polypeptides (comprising native crypticepitopes or optimized epitopes) can be enclosed in the kits instead ofsingle-epitope peptides.

In a particular embodiment of the invention, the kit is a vaccinationkit, wherein said first (native) and second (cognate optimized) peptidesare in separate vaccination doses. In a preferred embodiment, thevaccination kit comprises 2 or 3 doses of optimized peptide, and 3, 4, 5or 6 doses of native peptide. A particular vaccination kit according tothe invention is adapted for the first vaccination sequence of 6injections, and comprises 2 or 3 doses of optimized peptide, and 4 or 3doses of native peptide. In case of long-lasting diseases, it ispreferable to maintain the level of immunity obtained after thisprimo-vaccination, by regular recalls. This can be done, for example, byinjections performed every 1.5 to 6 months. Therefore, complementarykits, comprising at least 2 doses, and up to 40 or 50 doses of nativepeptide, are also part of the present invention. Alternatively, thevaccination kit can comprise 2 to 3 doses of optimized peptide, and 3 to40 or up to 50 doses of native peptide. Of course, said native andoptimized peptides present in the kit are as described above.

Each dose comprises between 0.5 and 10 mg of peptide, preferably from 1to 5 mg, or between 1 and 20 mg of polypeptide. In a preferredembodiment, each dose is formulated for subcutaneous injection. Forexample, each dose can be formulated in 0.3 to 1.5 ml of an emulsion ofaqueous solution emulsified with Montanide, used as an adjuvant. Theskilled artisan can choose any other adjuvant(s) in place of (or inaddition to) Montanide. In a particular embodiment, the doses are in theform of an aqueous solution. Alternatively, the doses can be in the formof a lyophilized peptide, for extemporaneous preparation of the liquidsolution to be injected. Other possible components of said kits are oneor several adjuvants, to be added to the peptide compositions beforeadministration, and a notice describing how to use said kits.

The invention is further illustrated by the following figures andexamples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Immunogenicity of HLA-B*0702 restricted peptides. CTL weretested against RMA-B7 targets loaded with peptide as indicated.

FIG. 2: Immunogenicity of optimized HLA-B*0702 cryptic peptides. CTLwere tested against RMA-B7 targets loaded with peptide as indicated.

FIG. 3: In vivo immunogenicity of optimized HLA-B*0702 Her2/neu_(1069L9)(A) and Her2/neu₁₀₆₉ (B) peptides in HLA-B*0702 transgenic mice. CTLwere tested against RMA-B7 targets loaded with peptide as indicated. CTLpopulation induced was diluted 3 (1), 10 (2), 30 (3) and 100 (4) fold.

FIG. 4: TERT₄ induces TERT specific CTL in HLA-B7 mice and in healthydonors. (A) TERT₄ immunogenicity in HLA-B*0702 transgenic mice. CTL weretested against RMA-B7 targets loaded with decreasing doses of TERT₄peptide. (B) Recognition of endogenous TERT by TERT₄ specific murineCTL. CTL were tested against COS cells transfected with HLA-B*0702 andTERT as indicated. (C) Induction of TERT₄ specific human CTL. CTL weretested against T2-B7 targets loaded with TERT₄ (▪) or an irrelevant ()peptide using the Effector/Target ratio as indicated (left graph), andagainst the HLA-B*0702 positive TERT positive SK-MES-1 (▪), HBL-100 ()and the HLA-B*0702 negative TERT positive SW-480 (□), HSS (◯) humantumor cell lines (right graph).

FIG. 5: Recognition of endogenous TERT by TERT₄₄₄ specific murine CTL.(A) CTL were tested against RMA-B7 targets loaded with decreasing dosesof TERT₄₄₄ or TERT_(444A1) peptides as indicated. (B) CTL were testedagainst COS cells transfected with HLA-B*0702 and/or TERT as indicated.

FIG. 6: Induction of TERT_(444A1) specific human CTL. CTL were testedagainst T2-B7 targets loaded with peptides as indicated. CTL maximalactivation is obtained by PMA/ionomycin treatment.

DETAILED DESCRIPTION

The examples have been performed using the following materials andmethods:

Transgenic Mice. The HLA-B7 H-2 class-I knockout mice were previouslydescribed (Rohrlich et al., 2003).

Cells. HLA-B*0702 transfected murine RMA-B7 and human T2-B7 cells werepreviously described (Rohrlich et al., 2003). COS-7 and WEHI-164 clone13 cells were provided by F. Jotereau (INSERM 463, Nantes, France). TheHLA-B*0702 positive SK-MES-1 (lung cancer), HBL-100 (breast cancer), andthe HLA-B*0702 negative SW-480 (colon cancer) and HSS (myeloma) celllines were used as targets of human CTL. All cell lines were grown inFCS 10% supplemented RPMI1640 culture medium.

Peptides and Plasmids. Peptides were synthesized by Epytop (Nimes,France). HLA-B*0702 plasmid was provided by Dr. Lemonnier (InstitutPasteur, Paris, France) (Rohrlich et al., 2003) and TERT plasmid wasprovided by Dr Weinberg (MIT, Boston, Mass.) (Meyerson et al, 1997).

Measurement of Peptide Relative Affinity to HLA-B*0702. The protocolused has been described previously (Rohrlich et al., 2003). Briefly,T2-B7 cells were incubated at 37° C. for 16 hours with peptidesconcentrations ranging from 100 μM to 0.1 μM, and then stained with ME-1monoclonal antibody (mAb) to quantify the surface expression ofHLA-B*0702. For each peptide concentration, the HLA-B*0702 specificstaining was calculated as the percentage of staining obtained with 100μM of the reference peptide CMV₂₆₅₋₂₇₄ (R10V; RPHERNGFTV, SEQ ID NO: 9).The relative affinity (RA) was determined as: RA=(Concentration of eachpeptide that induces 20% of HLA-B*0702-expression/Concentration of thereference peptide that induces 20% of HLA-B*0702 expression).

CTL Induction in vivo in HLA-B*0702 Transgenic Mice. Mice were injectedsubcutaneously with 100 μg of peptide emulsified in Incomplete Freund'sAdjuvant (IFA) in the presence of 150 μg of the I-A^(b) restrictedHBVcore₁₂₈ T helper epitope (TPPAYRPPNAPIL, SEQ ID NO: 10). After 11days, 5×10⁷ spleen cells were stimulated in vitro with peptide (10 μM).On day 6 of culture, the bulk responder populations were tested forspecific cytotoxicity.

Peptide Processing Assay on COS-7 Transfected Cells. 2.2×10⁴ simianCOS-7 cells were plated in flat-bottomed 96-well plates in DMEM+10% FCS,in triplicate for each condition. Eighteen hours later, cells weretransfected with 100 ng of each DNA plasmid with DEAE Dextran. After 4hours, PBS+10% DMSO was added for 2 minutes. Transfected COS cells wereincubated in DMEM+10% FCS during 40 hours and then used to stimulatemurine CTL in a TNFα secretion assay.

TNFα Secretion Assay. Transfected COS-7 cells at day 4 were suspended in500 of RPMI+10% FCS and used as stimulating cells. 5×10⁴ murine T cellswere then added in 50 μl RPMI 10% FCS and incubated for 6 hours. Eachcondition was tested in triplicate. 50 μl of the supernatant wascollected to measure TNFα. Standard dilutions were prepared in 50 μlwith final doses of TNFα ranging from 104 to 0 pg/ml. On both thesupernatants and the standard dilutions, 3×10⁴ TNFα sensitiveWEHI-164c13 cells in 50 μl were added. They were incubated for 16 h at37° C. Inhibition of cell proliferation was evaluated by the MTTcolorimetric method (Espevik and Nissen-Meyer, 1986).

Generation of CTL from human PBMC. PBMC were collected by leukapheresisfrom healthy HLA-B*0702 volunteers. Dendritic cells (DC) were producedfrom adherent cells cultured for seven days (2×10⁶ cells/ml) in thepresence of 500 IU/ml GM-CSF and 500 IU/ml IL-4 (R&D Systems,Minneapolis, Minn.) in complete medium (RPMI-1640 supplemented with 10%heat inactivated human AB serum, 2 μM L-Glutamine and antibiotics). Onday seven, DC were pulsed with 10 μM peptides for 2 hrs; maturationagents Poly I:C (Sigma, Oakville, Canada) at 100 ng/ml and anti-CD40 mAb(clone G28-5, ATCC, Manassas, Va.) at 2 μg/ml were added in the cultureand DCs were incubated at 37° C. overnight or up to 48 hours. Mature DCwere then irradiated (3500 rads). CD8+ cells were purified by positiveselection with CD8 MicroBeads (Miltenyi Biotec, Auburn, Calif.)according to the manufacturer's instructions. 2×10⁵ CD8⁺ cells+6×10⁴CD8⁻ cells were stimulated with 2×10⁴ peptide pulsed DC in completeculture medium supplemented with 1000 IU/ml IL-6 and 5 IU/ml IL-12 (R&DSystems, Minneapolis, Minn.) in round-bottomed 96 well plates. From dayseven, cultures were weekly restimulated with peptide-loaded DC in thepresence of 20 IU/ml IL-2 (Proleukin, Chiron Corp., Emeryville, Calif.)and 10 ng/ml IL-7 (R&D Systems, Minneapolis, Minn.). After the third invitro restimulation, bulk cell cultures were tested for cytotoxicity(TERT₄) or for IFNγ intracellular staining (TERT_(444A1)).

Cytotoxic assay. Targets were labelled with 100 μCi of Cr⁵¹ for 60 min,plated in 96-well V-bottomed plates (3×10³ cell/well in 100 μL of RPMI1640 medium) and, when necessary, pulsed with peptides (1 μM) at 37° C.for 2 hours. Effectors were then added in the wells and incubated at 37°C. for 4 hours. Percentage of specific lysis was determined as: %Lysis=(Experimental Release−Spontaneous Release)/(MaximalRelease−Spontaneous Release)×100.

IFNγ intracellular staining. T cells (10⁵) were incubated with 2×10⁵ T2cells loaded with stimulating peptide in the presence of 20 μg/mlBrefeldin-A (Sigma, Oakville, Canada). Six hours later they were washed,stained with r-phycoerythrin-conjugated anti-CD8 antibody (CaltagLaboratories, Burlingame, Calif., USA) in PBS for 25 min at 4° C.,washed again, and fixed with 4% PFA. The cells were then permeabilizedwith PBS, 0.5% BSA, 0.2% saponin (Sigma, Oakville, Canada), and labeledwith an allophycocyanin-conjugated anti-IFNγ mAb (PharMingen,Mississauga, Canada) for 25 min at 4° C. before analysis with aFACSCalibur® flow cytometer.

Examples Example 1: Affinity of Peptides

Eight peptides with the HLA-B*0702 specific anchor motifs, i.e. P2 andpreferentially L/V at C-terminal position (Sidney et al., 1996)belonging to Hsp70 (Hsp70₁₁₅, Hsp70₁₃₇, Hsp70₃₉₇), TERT (TERT₄ andTERT₄₄₄), and MAGE-A (MAGE-A_(121.1), MAGE-A_(121.2) and MAGE-A_(121.4))antigens were tested for binding to the HLA-B*0702 molecule. Only TERT₄bound to HLA-B*0702 with a high affinity, the remaining seven peptideswere very weak or non binders (Table II). This demonstrates that thepresence of anchor motifs is not sufficient to ensure a high bindingaffinity to HLA-B*0702. Given their low affinity, peptides Hsp70₁₁₅,Hsp70₁₃₇, Hsp70₃₉₇, TERT₄₄₄, MAGE-A_(121.1), MAGE-A_(121.2),MAGE-A_(121.4), are considered cryptic peptides.

TABLE II HLA-B*0702 affinity of peptides SEQ ID Peptide Sequence RA NO:1 Hsp70 115 YPEEISSMVL >10 11 Hsp70 115A1 APEEISSMVL >10 12 2Hsp70 137 (10) YPVTNAVITV >10 13 3 Hsp70 397 APLSLGLET >10 14 4 TERT4APRCRAVRSL   0.74 15 5 TERT444 DPRRLVQLL >10  1 TERT444A1 APRRLVQLL  1.4  5 6 MAGE-A121.1 EPVTKAEML >10 16 MAGE-121.1/A1 APVTKAEML >10 17 7MAGE-A121.2 EPFTKAEML >10 18 8 MAGE-A121.4 EPITKAEIL >10 19

Example 2: Immunogenicity of Selected Peptides

The low affinity Hsp₁₃₇, Hsp₁₁₅, Hsp₃₉₇, TERT₄₄₄ and the high affinityTERT₄ peptides have been tested for their capacity to induce a specificCTL immune response in HLA-B*0702 transgenic mice. Only the highaffinity TERT₄ was immunogenic confirming that immunogenicity ofpeptides is strongly related to their affinity for HLA (FIG. 1).

Example 3: Enhancement of Affinity of Low Affinity Peptides

Since all these cryptic peptides had favourable primary anchor motifs,enhancement of their affinity is a prerequisite for them to beimmunogenic. It required the identification of unfavourable secondaryanchor motifs and their substitution with favourable motifs. Thesesubstitutions should however preserve the conformation of the peptidesegment that interacts with the TCR (position 4 to position 8). Theinterest was, therefore, focused on secondary anchor positions 1 and 3:aliphatic amino acids are favourable motifs at position 1 (Sidney,Southwood et al., 1996). However, peptides Hsp70₁₁₅ and Hsp70₁₃₇ thathave a Y (tyrosine) at position 1 are non binders. Moreover, thesubstitution of the amino acid at position 1 by an A (alanine) that isalso favourable at this position. (Parker et al, 1994) enhances theaffinity of the TERT₄₄₄ but not of the Hsp70₁₁₅ and the MAGE-A_(121.1)peptides (Table II). This indicates that the presence of favourableamino acids at position 1 and anchor positions 2 and 9/10 cannot ensureby itself a high binding affinity of all peptides. In the other hand,positively charged peptides (R/H/K) have been described to be favourableat position 3 (Sidney et al., 1996) and ten out of 26 identified tumorand HIV derived immunogenic peptides have an R/K/H at position 3 (TableIII).

TABLE III Tumor and HIV derived HLA-B*0702 restricted epitopes SEQ IDAntigen Sequence NO: Reference NY-ESO-1 APRGVRMAV 20 Slager et al, 2004ICE SPRWWPTCL 21 Ronsin et al., 1999 RAGE-1 SPSSNRIRNT 22Gaugler et al., 1996 RU2AS LPRWPPPQL 23 Van Den Eynde et al., 1999RBAF500 RPHVPESAF 24 Lennerz et al., 2005 SSX2 fusion protein QPRYGYDQIM25 Worley et al, 2001 HIVp17 RPGGKKRYKL 26 HIV Molecular ImmunologyHIVp24 SPRTLNAWV 27 Database (Operated by Los HIVp24 HPVHAGPIA 28Alamos National Security, LLC, HIVp24 PPIPVGEIY 29for the U.S. Department of HIVp24 GPGHKARVL 30 Energy's National NuclearHIV-RT SPIETVPVKL 31 Security Administration) HIV-RT GPKVKQWPLT 32HIV-RT SPAIFQSSM 33 HIV-RT IPLTEEAEL 34 HIV-RT QPDKSESELV 35 HIV-VifHPRISSEVHI 36 HIV-Vif KPPLPSVKKL 37 HIV-Vif FPRTWLHGL 38 HIVgp160KPCVKLTPLC 39 HIVgp160 KVVSTQLLL 40 HIVgp160 RPWNNTRKSI 41 HIVgp160IPRRIRQGL 42 HIVnef FPVTPQVPL 43 HIVnef TPQVPLRPM 44

According to all these observations, peptides with the sequenceAPX₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁ (SEQ ID NO: 61) should have a high affinity forHLA-B*0702. This is confirmed by results shown in Table IV. All eighteenpeptides with the above cited sequence had a high affinity and/or wereimmunogenic in HLA-B*0702 transgenic mice.

TABLE IV Affinity and immunogenicity of APX₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁HLA-B*0702 restricted peptides. - = RA > 10, + = 1 < RA < 10, ++ = RA <1, Immunogenicity was tested as described in Exemple in 2. +: meansthat a specific immunoresponse was generated atleast one HLA-B*0702 transgenic mice, ND: Not Determined SequenceSEQ ID NO: RA Immunogenicity APRRLVQLL  5 + + APRSPLAPL  6 ++ +APKANKEIL  7 ND + APKHSDCLA  8 ND + APRCRAVRSL 15 + + APRMHCAVDL 45 ++ +APRVSIRLPL 46 ++ ND APREYVNAL 47 + + APRGVPQIEL 48 ND + APRALVETL 49 + +APRMPEAAL 50 ND + APRRRLGCEL 51 + + APRPWTPCL 52 + + APRASSPLL 53 ND +APRQLGREL 54 ND + APREISSMVL 55 + + APRSLGLEL 56 ++ + APRTKAEML 57 + +

Example 4: In Vivo Immunogenicity of Peptides with Enhanced Affinity andRecognition of the Native Counterpart

HLA-B7 transgenic mice were vaccinated with the selected peptides, andeleven days later, their spleen cells were in vitro stimulated with thepeptide.

In this context, Hsp70₁₁₅, Hsp70₃₉₇ and TERT₄₄₄, were therefore modifiedat position 1 (substitution of the amino acid by an A) and/or position 3(substitution of the amino acid by an R). For peptide Hsp70₃₉₇ anadditional modification at C-terminal position (substitution of the T byan L) has been introduced. Modified peptides i.e. Hsp70_(115A1R3) (SEQID NO: 55), Hsp70_(397R3L9) (SEQ ID NO: 56), TERT_(444A1) (SEQ ID NO: 5)exhibited a strong affinity for HLA-B*0702 (Table IV) and induced animmune response in the majority of vaccinated mice (FIG. 2). However,for all peptides but TERT_(444A1), generated CTL recognized theoptimized peptide but not the corresponding native peptide (FIG. 2).This strongly suggests that substitution of the amino acid at position 3by an R may change the conformation of the peptide segment thatinteracts with the TCR and guarantees TCR cross-recognition.

Since a) all tested peptides with APX₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁ have a highaffinity and are immunogenic (Table IV and FIG. 1, 2) and b)substitution of the amino acid at position 3 by an R may break thecross-recognition of the native peptide, the inventors selected nativepeptides having a P and R at positions 2 and 3 respectively, and theysubstituted the amino acid at position 1 by an A if the last amino acidwas favourable (L, A, I, V or M). Given the high importance of position3 in both affinity and CTL recognition of HLA-B*0702 restricted peptidesinventors selected peptides with the sequence X₁PX₃ (wherein X₁ is anyamino acid and X₃ is K, R, H or M; these amino acids have been describedas being favourable residues at position 3) and a favourable amino acid(A/I/L/V) at C-terminal position. Peptides with this sequence and lowaffinity for HLA-B*0702 have been modified by substitution of the firstresidue by an A. This is the case of TERT₄₄₄, Her-2/neu₇₆₀ andHer-2/neu₂₄₆. Inventors also selected peptides with the sequence APX₃(wherein X₃ is K, R, H or M) and a non favourable residue at C-terminalposition (i.e., an amino acid other than L, A, I, V or M). Peptides withthis sequence and low affinity for HLA-B*0702 have been modified bysubstituting residue at C-terminal position with a L. This is the caseof Her-2/neu₁₀₆₉. All these modified peptides had a strong affinity forHLA-B*0702.

Example 5: Immunogenicity of Optimized Peptides and Cross-Recognition ofthe Native Counterpart

Native Her2/neu₂₄₆, Her2/neu₇₆₀, Her2/neu₁₀₆₉ and TERT₄₄₄ peptides werenot immunogenic, whereas the optimized peptides were immunogenic inHLA-B*0702 transgenic mice. Moreover, CTL induced by all these optimizedpeptides were able to cross-react with the corresponding native peptide(FIG. 3 and Table V).

TABLE VImmunogenicity of native and optimized HLA-B*0702 restricted peptides. +for immunogenicity or native peptide cross recognition means thatthe peptide induced a specific response in at least one HLA-B*0702transgenic mouse, able to recognized the corresponding native peptide.Corresponding native SEQ ID Immuno- peptide Peptide Sequence NO:genicity cross-reconnaissance TERT₄₄₄ DPRRLVQLL 1 - TERT_(444A1)APRRLVQLL 5 + + Her2/neu₇₆₀ SPKANKEIL 3 - Her2/neu_(760A1) APKANKEIL7 + + Her2/neu₂₄₆ GPKHSDCLA 4 - Her2/neu_(246A1) APKHSDCLA 8 + +Her2/neu₁₀₆₉ APRSPLAPS 2 - Her2/neu_(1069L9) APRSPLAPL 6 + +

In conclusion, the inventors have described a method to optimizeimmunogenicity (and also affinity) of HLA-B*0702 restricted crypticpeptides. It consists in a) substituting the residue at position 1 withan A in all peptides comprising the sequence X₁PX₃ (wherein X₁ is anyamino acid except A and X₃ is R or K or H or M), a favourable amino acidat C-terminal position (i.e., L or A or I or V or M), and a low affinityfor HLA-B*0702, or b) substituting the residue at C-terminal positionwith a L in peptides comprising the sequence APX₃ (X₃ being defined asabove), a non favourable residue at C-terminal position (i.e., an aminoacid other that L or A or I or V or M), and a low affinity forHLA-B*0702.

Example 6: TERT₄ Immunodominant Peptide Induces TERT Specific CTL

HLA-B7 transgenic mice were then immunized with the TERT₄ (SEQ ID NO:15) and eleven days later their spleen cells were in vitro stimulatedwith the peptide. Generated CTL killed RMA-B7 targets loaded withdecreasing concentrations of TERT₄ peptide (FIG. 4A). The half maximallysis of TERT₄ loaded targets was obtained with 1.5 nM (FIG. 4A). CTLwere then tested for their capacity to recognize COS-7 cells expressingHLA-B*0702 and endogenous TERT. Results presented in FIG. 4B show thatCTL recognized COS-7 cells transfected with both HLA-B*0702 and TERT butnot COS-7 cells transfected with either HLA-B*0702 or TERT,demonstrating that TERT₄ dominant peptide is an HLA-B*0702 restrictedepitope naturally processed from endogenous TERT.

Moreover, CD8 cells from healthy donors were in vitro stimulated withautologous dendritic cells loaded with TERT₄ peptide. After fourstimulations, CTL were tested for cytotoxicity against TERT₄ loadedT2-B7 targets. Three donors were tested and CTL were induced in two ofthem. Results from one responding donor are presented in FIG. 4C. CTLkilled T2-B7 targets presenting TERT₄ but not T2-B7 cells presenting theirrelevant Nef peptide (left graph). Interestingly, CTL killed theHLA-B*0702 TERT+SK-MES-1 and HBL-100 but not the HLA-B*0702-TERT+SW-480and HSS human tumor cell lines confirming the HLA-B*0702 restrictedpresentation and the endogenous processing of the TERT₄ epitope (rightgraph).

Example 7: CTL Induced by TERT_(444A1) Peptide Recognize Endogenous TERT

TERT_(444A1) (SEQ ID NO: 5) was tested for its ability to induce CTLable to recognize endogenous TERT and to induce CTL in healthy donors(Example 6). HLA-B*0702 transgenic mice were then immunized with theTERT_(444A1) and eleven days later their spleen cells were in vitrostimulated with the native TERT₄₄₄ peptide (SEQ ID NO: 1). Generated CTLkilled RMA-B7 targets loaded with decreasing concentrations ofTERT_(444A1) and TERT₄₄₄ peptides. The half maximal lysis of TERT₄₄₄loaded and TERT_(444A1) loaded targets was obtained with 5.5 nM and 1 nMrespectively (FIG. 5A). CTL were then tested for their capacity torecognize COS-7 cells expressing HLA-B*0702 and endogenous TERT. Resultspresented in FIG. 5B show that CTL recognized COS-7 cells transfectedwith both HLA-B*0702 and TERT but not COS-7 cells transfected witheither HLA-B*0702 or TERT demonstrating that TERT₄₄₄ is an HLA-B*0702restricted cryptic epitope naturally processed from endogenous TERT.

Example 8: TERT_(444A1) Stimulates CTL from Healthy Donors

CD8 cells from healthy donors were in vitro stimulated with autologousdendritic cells loaded with TERT_(444A1) peptide. After fourstimulations, proliferating cells were divided into 4 pools. Each poolwas then tested for intracellular IFNg production upon stimulation withT2-B7 cells loaded with optimized TERT_(444A1) or native TERT₄₄₄.Results from D5609 responding donor are presented in FIG. 6. IFNgproducing CTL were detected in pools 2 and 4 after stimulation witheither TERT₄₄₄ or TERT_(444A1) loaded T2B7 cells (FIG. 6).

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1-20. (canceled)
 21. An immunogenic HLA-B*0702-restricted epitope havingthe sequence APRRLVQLL (SEQ ID NO: 5).
 22. A chimeric polypeptide,comprising the peptide DPRRLVQLL (SEQ ID NO: 1) and one, two or moreHLA-B*0702-restricted cryptic epitopes selected from the groupconsisting of GPKHSDCLA (SEQ ID NO: 4), SPKANKEIL (SEQ ID NO: 3),APRSPLAPS (SEQ ID NO: 2), and DPRRLVQLL (SEQ ID NO: 1).
 23. A chimericpolypeptide, comprising SEQ ID No: 5 and one, two or more immunogenicHLA-B*0702-restricted epitopes selected from the group consisting ofAPKHSDCLA (SEQ ID NO: 8), APKANKEIL (SEQ ID NO: 7), APRSPLAPL (SEQ IDNO: 6), and APRRLVQLL (SEQ ID NO: 5).
 24. A pharmaceutical compositioncomprising at least, as an active principle, an immunogenicHLA-B*0702-restricted epitope polypeptide according to claim
 21. 25. Apharmaceutical composition comprising at least, as an active principle,a chimeric polypeptide according to claim
 22. 26. A pharmaceuticalcomposition comprising at least, as an active principle, a chimericpolypeptide according to claim
 23. 27. The pharmaceutical composition ofany one of claims 24 to 26, which is a vaccine.
 28. A kit of partscomprising, in separate formulations, a first chimeric polypeptideaccording to claim 22, and a second chimeric polypeptide according toclaim
 23. 29. The kit according to claim 28, which is a vaccination kit,wherein said first and second chimeric polypeptides are in separatevaccination doses.
 30. The vaccination kit according to claim 29, whichcomprises 2 or 3 doses of second chimeric polypeptide, and 3, 4, 5, 6 orup to 50 doses of first chimeric polypeptide.
 31. The vaccination kitaccording to claim 29, wherein each dose comprises 1 to 20 mg ofchimeric polypeptide.
 32. The vaccination kit according to claim 29,wherein the vaccination doses are formulated for subcutaneous injection.33. A kit of parts comprising, in separate formulations, a chimericpolypeptide according to claim 22 and an immunogenic peptide accordingto claim
 21. 34. The kit according to claim 33, which is a vaccinationkit, wherein said chimeric polypeptide and immunogenic peptide are inseparate vaccination doses.
 35. The vaccination kit according to claim33, which comprises 2 or 3 doses of immunogenic peptide, and 3, 4, 5, 6or up to 50 doses of chimeric polypeptide.
 36. The vaccination kitaccording to claim 35, wherein each dose comprises 1 to 20 mg ofchimeric polypeptide or of immunogenic peptide.
 37. The vaccination kitaccording to claim 33, wherein the vaccination doses are formulated forsubcutaneous injection.